The cleavage mechanism utilized for expression of the polyprotein NS3- NS4A-NS4B-NS5 domain of dengue virus was studied with the aim of elucidating the functional activities of these dengue virus proteins. For this purpose, recombinant vaccinia viruses v(NS2B-NS3-NS4A-NS4B- NS5), v(NS3-NS4A-NS4B-NS5), v(NS4A-NS4B-NS5), and v(NS4B-NS5) were constructed. These recombinants were used to infect cells and the labelled lysates were analyzed by NS3 or NS5 specific antiserum. Our findings indicated that NS2B is required for processing of the downstream nonstructural proteins. In the presence of NS2B supplied in trans, polyprotein NS3-NS4A-NS4B-NS5 was cleaved at the NS3-NS4A junction although less efficiently than at the NS4B-NS5 junction. The flavivirus NS4A-NS4B cleavage junction follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 segment was properly cleaved at the NS4A-NS4B junction in the absence of other dengue viral functions such as the NS1-NS2A and NS2B-NS3 protease systems. However, v(NS3-NS4A-NS4B-NS5) expressed only the uncleaved polyprotein precursor. Thus, cleavage at the NS3-NS4A junction appears to be a prerequisite for cleavage at the downstream junction to take place. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as NS3-NS4A-NS4B-NS5, NS4A-NS4B-NS5 or NS4B-NS5, produced properly cleaved NS5 when used for coinfection with v(NS2B-NS3 30%), or with v(NS2B) plus v(NS3). These results indicated that cleavage at the NS4B-NS5 junction of the polyprotein is mediated by NS2B and NS3 in trans.